Targeted PPAR{gamma} deficiency in alveolar macrophages disrupts surfactant catabolism

Abstract

Surfactant accumulates in alveolar macrophages of granulocyte-macrophage colony-stimulating factor (GM-CSF) knockout (KO) mice and pulmonary alveolar proteinosis (PAP) patients with a functional loss of GM-CSF resulting from neutralizing anti-GM-CSF antibody. Alveolar macrophages from PAP patients and GM-CSF KO mice are de-ficient in peroxisome proliferator-activated receptor-gamma (PPARgamma) and ATP-binding cassette (ABC) lipid transporter ABCG1. Previous studies have demonstrated that GM-CSF induces PPARgamma. We therefore hypothesized that PPARgamma promotes surfactant catabolism through regulation of ABCG1. To address this hypothesis, macrophage-specific PPARgamma (MacPPARgamma) knockout mice were utilized. MacPPARgamma KO mice develop foamy, lipid-engorged Oil Red O positive alveolar macrophages. Lipid analyses revealed significant increases in the cholesterol and phospholipid contents of MacPPARgamma KO alveolar macrophages and extracellular bronchoalveolar lavage (BAL)-derived fluids. MacPPARgamma KO alveolar macrophages showed decreased expression of ABCG1 and a deficiency in ABCG1-mediated cholesterol efflux to HDL. Lipid metabolism may also be regulated by liver X receptor (LXR)-ABCA1 pathways. Interestingly, ABCA1 and LXRbeta expression were elevated, indicating that this pathway is not sufficient to prevent surfactant accumulation in alveolar macrophages. These results suggest that PPARgamma mediates a critical role in surfactant homeostasis through the regulation of ABCG1.

Fig. 1.

PPARγ deficiency results in dysregulation of lipid metabolism in alveolar macrophages. (A) Marked Oil Red O staining of alveolar macrophages from MacPPARγ KO indicates neutral lipid accumulation compared with wild-type (n = 3). (B) ABCG1 is decreased whereas ABCA1 expression is enhanced in MacPPARγ KO compared with wild-type as measured by RT-PCR (n = 6). Data represent mean ± SEM. (C) ABCG1 protein is decreased and (D) ABCA1 protein is increased in MacPPARγ KO alveolar macrophages as shown in a representative immunoblot from one of two experiments. The numbers above the bands refer to sets of pooled BAL cells from each genotype. The relative ratios of the ABCG1 and ABCA1 to actin are indicated. ABC, ATP binding cassette; KO, knockout; MacPPARγ, macrophage-specific PPARγ; WT, wild-type.

Fig. 2.

Surfactant lipids accumulate in the lungs of MacPPARγ KO mice. (A–B) The free cholesterol content of MacPPARγ KO alveolar macrophages (n = 3 sets) and BAL fluid (n = 5) is increased. Total and free cholesterol were measured and cholesteryl ester was determined by subtraction (51). (C–D) The phospholipid content is also increased in the alveolar macrophages (n = 4 sets) and BAL fluid (n = 11) of MacPPARγ KO mice. Data represent mean ± SEM. BAL, bronchoalveolar lavage; KO, knockout; MacPPARγ, macrophage-specific PPARγ; WT, wild-type.

Fig. 3.

PPARγ deficiency results in decreased cholesterol efflux to HDL from alveolar macrophages. The efflux of ³H labeled cholesterol was measured in MacPPARγ KO alveolar macrophages and compared with wild type (n = 3). Apo, apolipoprotein; KO, knockout; MacPPARγ, macrophage-specific PPARγ; WT, wild-type.

Fig. 4.

PPARγ deficiency results in dysregulated LXRα and LXRβ expression. (A) Gene expression of LXRα and LXRβ in BAL cells from wild-type (n = 4) and MacPPARγ KO mice (n = 6) were analyzed by RT-PCR. LXRα mRNA expression is decreased in MacPPARγ KO alveolar macrophages while LXRβ is increased. (B) CYP27A1 and ApoE mRNA expression are increased in MacPPARγ KO alveolar macrophages (n = 6) compared with wild type (n = 5). Data represent mean ± SEM. Apo, apolipoprotein; KO, knockout; LXR, liver X receptor; MacPPARγ, macrophage-specific PPARγ; WT, wild-type.